BIOLOGIA PLANTARUM 56 (4): 620-627, 2012 DOI: 10.1007/s10535-012-0256-2

A pattern of unique embryogenesis occurring via in Carya cathayensis

B. ZHANG1,2a, Z.J. WANG1a, S.H. JIN1a, G.H. XIA1, Y.J. HUANG1 and J.Q. HUANG1*

School of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, P.R. China1 Bureau of Agricultural Economics of Haiyan, Haiyan 314300, P.R. China2

Abstract

Apomixis represents an alteration of classical sexual plant reproduction to produce that have essentially clonal . In this report, hickory (Carya cathayensis Sarg.), which is an important oil tree, is identified as a new apomictic species. The ovary has a chamber containing one that is unitegmic and orthotropous. Embryological investigations indicated that the developmental pattern of sac formation is typical polygonum-type. Zygote embryos were not found during numerous histological investigations, and the embryo originated from nucellar cells. Nucellar embryo initials were found both at the micropylar and chalazal ends of the embryo sac, but the mature embryo developed only at the nucellar beak region. The mass of the nucellar embryo initial at the nucellar beak region developed into a nucellar embryo or split into two nucellar proembryos. The later development of the nucellar embryo was similar to the zygotic embryo and progressed from globular embryo to heart-shape embryo and to cotyledon embryo. Additional key words: adventitious embryony, female , hickory, nucellar embryo.

Introduction

The term apomixis was first introduced by Winkler 3) two gametophytic types diplospory and apospory (1908) to refer to the “substitution of sexual reproduction (Peggy 2006, Koltunow 1993). Adventitious embryos can by an asexual multiplication process without nucleus and arise from two different tissues of the mature ovule, cell fusion”. Apomixis in flowering plants is the asexual namely the nucellus and the inner integument formation of directly from the maternal tissues of the (Lakshmanan and Ambegaokar 1984, Miles 2007). The ovule, avoiding and fertilization (Bicknell and form of the nucellus is common and this has been Koltunow 2004). Seeds resulting from apomixis are described in detail in , which is a model system for genetically identical to maternal plants, which is studying nucellar embryony. important for promoting the breeding process through Hickory (Carya cathayensis Sarg.), a member of the fixing hybrid vigor and arousing researchers’ interest family Juglandaceae, is an important oil tree of high (Hanna 1995, Koltunow et al. 1995a, Savidan 2000). economic value (Li 2003). In walnut, apomixis was Apomixis has been described in more than 400 flowering confirmed (Loiko 1990, Asadian and Pieber 2005), and plant species belonging to over 40 families (Carman later it was investigated in different cultivars using 1997). Among fruit tree species, only a few show this isoenzyme analysis (San and Dumanoğlu 2006, Wu et al. phenomenon and these fruits generally propagate 2006). The mechanisms of apomixis in walnut have been vegetatively, including citrus and mango (Mo et al. 2005). reported as adventitious embryony (Valdiviesso 1990), Apomixis can be divided broadly into three types: apospory (Terziiski and Stefanova 1990) or diplospory 1) adventitious embryony, 2) a sporophytic type, or (Sartorius and Stosser 1991). Sartorius and Stösser (1997)

⎯⎯⎯⎯

Received 7 April 2011, accepted 7 October 2011. Abbreviations: AFLP - amplified fragment length polymorphism; DAP - days after pollination; FCSS - flow cytometric seed screening; SSR - simple sequence repeat. Acknowledgments: This study was supported by the National Natural Science Foundation of China (30872047, 31170584 and 31100229), the Zhejiang Provincial Natural Science Foundation (z307534 and Y3110311). a - These authors contributed equally to this work. * Corresponding author; fax: (+86) 571 63740809, e-mail: [email protected]

620 HICKORY IS A NEW APOMICTIC SPECIES and Chen et al. (2000) reported development of apomictic differences between the hybrids and parents were embryos from the in different walnut cultivars. analyzed. Differential bands were found between the Schanderl (1964) reported that the development of paternal and maternal parents, as well as between the apomictic embryos in walnut was diplosporic and not by paternal parent and the progeny, but there was no nucellar embryony. During our previous study with difference between the maternal parent and the progeny hickory (Wang et al. 2010), we observed a certain or amongst the progeny (Wang et al. 2010). In addition, proportion of polyembryonic seedlings (9.04 %). flower bud differentiation in hickory was studied in detail Furthermore, we conducted a hybrid experiment between (Huang et al. 2006, 2007). The aim of the present study hickory and pecan (C. illinoensis). The hybrids showed was to investigate the female gametophyte and embryo variations in fruit shape, pericarp colour and seed size development in hickory to understand the mechanisms of and mass. Using short-sequence repeat (SSR) and its reproductive processes. amplified fragment length polymorphism (AFLP),

Materials and methods

A 50-year-old adult hickory (Carya cathayensis Sarg.) August. All the collected materials were fixed and stored tree from Lin’an city, Zhejiang province (China) was in FAA (70 % ethanol + glacial acetic acid + form- chosen as the mother tree. The female flowers were aldehyde; 90:5:5). Fruits grew rapidly until mid-June. bagged when the stigma began to turn red on 11 May Thus, the shells of any large fruits that were collected 2010. During the pollination period, the female flowers were removed or the were directly isolated before were pollinated with mixed pollen from the mother tree fixing. Pistils and ovules were dehydrated in an ascending and surrounding trees on 17 May. One week later the series of ethanol, cleared in xylene and embedded in paper bags were removed once male inflorescences had paraffin. After trimming, paraffin blocks of tissue were faded. cut into 7 - 8 µm slices using a microtome (Leica When florets were visible (approximately 10 mm), RM2235, Leica Microsystems, Germany). Dry sections male flowers were collected every 4 d, and female were stained with safranin-fast green and iron flowers were collected at different developmental stages hematoxylin (Gao et al. 2010, Lin et al. 2010). according to the colour of the stigma. After pollination, Observations were carried out with a microscope female flowers and young fruits were collected every day (Olympus BX 60, Tokyo, Japan) and photographs were during the first 7 d, and then at 4-d intervals until mid- taken with the auto-photo system.

Results

Hickory is a monoecious tree with male flowers that are The ovary had a chamber containing one ovule that ternate aments and female flowers that form a terminal was unitegmic and orthotropous (Fig. 2A). In late April, spike of 3 - 4 florets. The floret is naked and without the ovule primordium was initiated in the mid-region of perianth. At the end of April, the floret was coated by the ovary, and then the integument primordium began to four bracts, which could be seen easily with the naked develop. The ovules had no funiculus and single eye (Fig. 1B). Afterwards, the bracts unfolded gradually integument. However, this integument did not completely with the development of the florets. During early May, cover the micropylar end of the nucellus and the the stigma appeared and then green circular spots were micropyle. Indeed, integument length was approximately presented (Fig. 1C). The pistil developed and extended half the length of the ovule during the mature embryo sac outwards and, due to the different growth rate of the pistil stage (Fig. 2B). tissues, it presented a dark-green rhombus after 2 - 3 d In early May, the mother cell, which was (Fig. 1D). The stigma colour changed from light red to different from surrounding nucellar cells, formed in the bright red to purplish red during the developmental stages middle of the nucellus. At this stage, stigmas of florets (Figs. 1E, 2H), and after it was pollinated, it quickly turned red starting from the central parts. Compared with turned dark (Fig. 1H). From mid-May to the mid-June the nucellar cells, the megaspore mother cell had a larger fruit size increased from 2.41 ± 0.07 mm on May 18 to nucleus and denser cytoplasm (Fig. 2C). Dyad (Fig. 2D) 9.24± 0.08 mm on June 24 and the average growth rate and tetrad formed after megasporocyte meiosis. At the was 0.18 mm per day. From June 24 to August 12, the tetrad stage, four aligned linearly along the fruits enlarged greatly and mean diameter increased to plane from micropylar to chalazal ends (Figs. 2E,F). 30.3 ± 0.12 mm. After that, the fruit diameter did not Then, the megaspore at the chalazal end became a increase up to harvest in early September (Figs. 1I,O). functional megaspore, while the others did not develop

621 B. ZHANG et al.

Fig. 1. Morphological observations of floret and fruit development. A - Female flower bud begins to differentiate at the apical end of the spring shoot at 17 April, bar = 0.5 mm. B - Florets coated with bracts become macroscopic by 4 May, bar = 1 mm. C - Female flower with three florets which are coated with four bracts at 9 May, bar = 6 mm. D - Naked florets with no perianth and rhombus- shape stigma which is not coated in bracts at 11 May, bar = 4 mm. E - Florets with light red stigmas at 13 May, bar = 5 mm. F - Florets with bright red stigmas at 16 May, bar = 4 mm. G - Florets with purplish red stigmas at 18 May, bar = 4 mm. H -Florets with dark purple stigmas at 5 DAP, bar = 5 mm. I - Young fruits at 10 DAP, bar = 2 mm. J - Young fruits at 26 DAP, bar = 3 mm. K - Young fruits at 30 DAP, bar = 3 mm. L - Young fruits at 54 DAP, bar = 6 mm. M - Young fruits at 80 DAP, bar = 5 mm. N - Young fruits at 87 DAP, bar = 5 mm. O - Young fruits at 95 DAP, bar = 7 mm. and gradually degenerated to remnant vestiges (Fig. 2F-H). embryo sac stages during pollination. The embryo sac Female flowers with bright red stigmas were at the tetrad was of the typical polygonum-type. stage. We observed histologically that the division of the The functional megaspore continued to develop and it primary nucleus took place initially at 5 d grew bigger (Fig. 2H). Concomitantly, the embryo sac after pollination (DAP; Fig 3A). The fusion of the two enlarged. At the two-nucleate embryo sac stage, the two male and the egg was not observed. At 10 DAP, nuclei moved toward opposite poles along the plane a few free nuclei, presumably originating from the between the micropylar to chalazal ends (Figs. 2I,J). The division of the primary endosperm nucleus, were detected stigmas were purplish red on female flowers when the in a vacuole-like compartment (Fig. 3B). These nuclei megagametophyte was at the four- and eight-nucleate were enclosed within cytoplasmic strands, which crossed stages. Nuclei continued to divide in the four-nucleate the embryo sac from the micropylar to the chalazal pole. embryo sac (Fig. 2K). The eight-nucleate embryo sac had Zygote embryos were not found at this stage. During the 4 nuclei at each pole, then one nucleus from each pole early stages of ovule growth, the free nuclei of the has migrated toward the center of the embryo sac endosperm were embedded in a strand of cytoplasm (Fig. 2L). When the floret stigma was dark purple, the along the outer margin of the central vacuole that was in mature embryo sac was formed with three antipodal cells contact with the nucellus (Fig. 3E). At 10 DAP, changes at the chalazal end and two polar nuclei and an egg were observed for some nucellus cells above the embryo apparatus at the micropylar end (Figs. 2M-O). The sac at the micropylar end. These cells contained a large development of female flowers is asynchronous. Stigmas nucleus or multiple nuclei, and these nuclei were of florets were of different colours during the pollination surrounded by saccular structures (Fig. 3C). Furthermore, period, but most were bright red and purplish red. Female there were also changes in the cells adjacent to the sexual flowers were mostly at the bi-nucleate or four-nucleate embryo sacs at the chalazal end, including a large

622 HICKORY IS A NEW APOMICTIC SPECIES

Fig. 2. Megasporogenesis and development of the female gametophyte in Carya cathayensis. A - The ovary has a chamber with an ovule that is unitegmic and orthotropous. B - The ovule is unitegmic, orthotropous and crassinucellate; i - integument, n - nucellus. C - Megaspore mother cell (MMC). D - Dyad. E - Four megaspores forming one linear tetrad. F - Three megaspores being dissolved at the micropylar end, while one is developing at the chalazal end. G - The second megaspore being dissolved at the chalazal end (os), two megaspores still being kept at the micropylar end and the one megaspore developing at the chalazal end. H - Three megaspores near the micropyle degenerate in succession, and the functional megaspore (ss) at the chalazal end. I and J - Bi-nucleate embryo sac with two nuclei distributed along the plane between the micropylar and chalazal ends. K - The four-nucleate embryo sac. L - The eight-nucleate embryo sac showing three antipodal cells (ant) and four nuclei at the chalazal end. M to O - Successive sections of a mature embryo sac; M - a synergid (sy) at the micropylar end and an antipodal (ant) at the chalazal end; N - the egg apparatus (ea) and two antipodals (ant); O - a synergid (sy) and a polar nuclei (pn). Bars: A = 200 µm; B = 100 µm; C - O = 20 µm.

623 B. ZHANG et al. cell nucleus and granular cytoplasm (Fig. 3D). These from the micropylar and chalazal end regions, and cells were isolated from the surrounding nucellar cells. nucellar cells at the chalazal end were associated with During endosperm development, the embryo sac loose structures (Fig. 3F). Endosperm was absent from developed gradually and the ovules enlarged. The some ovules (Figs. 3G-I). In these ovules, the embryo integument grew and coated the nucellus, which mostly sacs were narrower and nucellar embryo initials, remained only in the nucellar beak region (Fig. 3F). composed of cells with differing degrees of vacuoli- Degradation of the nucellus was observed during the zation, were found at the edges of the embryo sacs at the development and enlargement of the embryo sac in the chalazal end (Fig. 3G). Nucellar cells at the nucellar beak ovule at 26 DAP. The nucellus had been partly absorbed region showed differing degrees of vacuolization, and

624 HICKORY IS A NEW APOMICTIC SPECIES

Fig. 3. Nucellar embryony and development of the nucellar embryo and endosperm. A - Four free nuclei divided from the primary endosperm and the egg cell at the micropylar end of the embryo sac at 5 DAP, bar = 50 µm. B - Many free nuclei were encapsulated into the annular membrane around the central vacuole at 10 DAP, bar = 50 µm. C - Some nucellar cells elongated, enlarged and contained large and/or multiple cell nuclei with saccular structures surrounding the area above the sexual embryo sac at the micropylar end at 10 DAP, bar = 50 µm. D - Nucellar embryo initials with large cell nucleus and granular cytoplasm also existed at the chalazal end of embryo sac at 10 DAP, bar = 50 µm. E - Endosperm developed and free nuclei were encapsulated into the annular membrane around the central vacuole at 19 DAP, bar = 50 µm. F - The integument coat nucellar mostly only remained at the nucellar beak region; nucellar cells were absorbed gradually at 26 DAP, bar = 200 µm. G - A nucellar embryo initial developing at the chalazal end of embryo sac in an ovule without developing endosperm at 33 DAP, bar = 100 µm. H and I - Ovules lost endosperm at 33 DAP, nucellar cells at the beak region were large and vacuolization had occurred to differing degrees; the cells formed an irregularly shaped mass nucellar embryo initial, bar = 100 µm. J - The ovule enlarged greatly, especially at the chalazal end; the nucellus has been mostly absorbed except for the epidermal cells and at the nucellar beak region during late June (38 DAP), bar = 100 µm. K - Magnified nucellar beak region observed in J; nucellar cells at the nucellar beak region above the embryo sac were large and contained vacuoles to differing degrees, bar = 200 µm. L - The lower magnification on the left shows that the nucellus has been mostly absorbed except for the epidermal cells and at the nucellar beak region during late June (38 DAP), bar = 100 µm; the magnified nucellar beak region showing large cells with vacuolization to differing degrees that formed a mass nucellar embryo initial, bar = 200 µm. M - A block cell mass formed above the embryo sac at the micropylar end with the development of the whole nucellar embryo initial at June 29 (42 DAP), bar = 200 µm. N - Two block-like nucellar proembryos in an ovule, bar = 200 µm. O - Lower magnification on the left shows the whole ovule at July 3 (46 DAP), bar = 100 µm; the nucellar beak region magnified on the right shows the rod-shaped nucellar embryo, bar = 200 µm. P - Two rod-shaped nucellar embryos in the micropylar end, of which one was initiated from the whole mass nucellar embryo initial at the nucellar beak region, while the other was initiated from the epidermal cell in the micropylar end at July 3 (46 DAP), bar = 200 µm. Q - Globular embryo stage similar to the zygotic embryo at 51 DAP, bar = 200 µm. R - Heart-shape embryo stage similar to the zygotic embryo at 59 DAP, bar = 200 µm. S and T - Cotyledon embryo stage at 65 DAP, bar = 200 µm (es - embryo sac; en - endosperm; i - integument; n - nucellus; ni - nucellar embryo initial; nb - nucellar beak; ec - nucellar epidermal cell; ne - nucellar embryo. irregularly shaped embryos with dense cells were found (Figs. 3K,L). The nucellar embryo initial developed into at 33 DAP (Fig. 3H,I). This type of ovule was not found some small compact cells. One or more cell masses during later developmental stages. formed above the embryo sac at the micropylar end In the ovules that contained a developing endosperm, during the development of the nucellar embryo initial at the nucellar embryo initials were not found at the chalazal June 29 (42 DAP) (Fig. 3M,N). Cell masses were end of the embryo sac. Up to late June (38 DAP), the irregularly shaped during the early stages, and in some nucellus was absorbed, leaving mostly only epidermal ovules cell masses split into two nucellar proembryos cells and some nucellus at the nucellar beak region (Fig. 3O,P). Rod-shaped nucellar embryos formed during (Fig. 3J). From mid-June to mid-July, there was great the cell division (Fig. 3O,P). The later development of enlargement of the ovules, especially at the chalazal end. the nucellar embryo was similar to the zygotic embryo Except for the 1 - 2 layers of epidermal cells, nucellar and it underwent changes from globular stage (Fig. 3Q) cells at the nucellar beak region above the embryo sac to heart-shape stage (Fig. 3R) and, finally, to cotyledon were large and formed a mass nucellar embryo initial stage (Fig. 3S,T). with an obvious boundary with the surrounding cells

Discussion

Most sporophyte reproducing plants are diploid and embryo originates from an inner integument cell, is the polyembryonic. The zygotic and apomixis embryos only case of obligate apomixis in woody plants (Horn coexist in an ovule and can be distinguished easily 1940). The reasons behind the absence of sexual embryos according to their position in the ovule (Richards 2003). in hickory may be complex. Although germination and Female gametophyte development in hickory is normal, growth of the pollen tube on the stigma was observed, the but the zygotic embryo is absent in large numbers of process of pollen tubes growing through the tissue of the tissue cuttings and mature embryos all originate from the style and entering the embryo sac was difficult to follow nucellus. AFLP and SSR analyses of C. cathayensis × with the fixatives and stains used (data not shown). In C. illinoensis hybrids also showed that the progeny was walnut, the growth of pollen tubes can proceed through identical with the mother (Wang et al. 2010). Apomixis either the micropyle (porogamy) or the funiculus and in hickory is high and trends to be obligate, but chalazal tissues (chalazogamy) depending on the development of a sexual embryo cannot be excluded. developmental stage of the micropyle (Luza and Polito Most apomictic plant species are facultative for apomixis 1991). However, in this study, the single integument of and complete obligate apomixis is rare (Asker and Jerling hickory can not envelop the ovule in the mature 1992). Garcinia mangostana, whose single adventitious embryonic sac phase. Synergids also play an important

625 B. ZHANG et al. role in the guidance of pollen tubes (Higashiyama et al. in Tabebuia ochracea (initial cell divides and forms 2001, Higashiyama 2002). From pollen germination to tubular shape nucellar embryo that is composed of apical fertilization, many factors may lead to fertilization failure cell and basal cell) (Costa et al. 2004). of the egg cell. In many angiosperms, a 2:1 maternal:paternal ratio in Initial cells of nucellar embryo are evident histolo- the endosperm is optimal for seed development, and this gically in the ovules of flowers prior to anthesis. In vitro results from genomic imprinting (Vinkenoog et al. 2003). culture of ovules from flowers at different prepollination In the vast majority (ca. 90 %; Mogie 1992) of apomictic stages showed that embryos could develop from ovules plants production of viable seed depends on the presence cultured as early as the binucleate stage of megagameto- of pollen, i.e., fertilization of the polar nuclei is still genesis in which nucellar initial cells were absent required for normal development of the endosperm histologically (Koltunow et al. 1995b). Some nucellar (pseudogamy). Only a few apomictic taxa, mainly cells showed changes in 10 DAP ovules, specifically cells occurring in the Asteraceae and including genus were enlarged and elongated, contained a large nucleus or Taraxacum and Hieracium, can develop endosperm multiple nuclei, and these nuclei were surrounded by autonomously. Moreover, Commiphora wighti, can form saccular tissues. The morphology of these cells is sexual and apomixis embryos simultaneously and different from the nucellar embryo initial cells in Citrus. develop endosperm autonomously (Gupta et al. 1996). The function of these changed nucellar cells is unknown Genomic imprinting in these autonomous apomicts is but closely related to the degradation of nucellar cells and probably bypassed (Curtis and Grossniklaus 2007). the formation of embryogenic cells later at the nucellar Endosperm develops in most ovules and is absent in a beak region. The initial cells of nucellar embryo were few young fruits that later abort. This indicates that the distributed mainly above the embryo sac at the micro- occurrence of the nucellar embryo does not depend on pylar end and adjacent to the sexual embryo sac at the endosperm development. In order to determine the source chalazal end. However, the mature nucellar embryo was of the endosperm further, another study was conducted to found only at the nucellar beak region, which might be bag and isolate more than 100 female flowers before the due to the inhibition of the developing endosperm as stigmas turned red. We found that young fruits dropped reported previously in Citrus (Wakana and Uemoto 1987, completely within 30 d, and it seemed that pollination 1988, Koltunow 1995b). There is strong evidence that the was necessary for fruit setting (unpublished observation). nucellar embryo initial only existed in ovules that showed Endosperm is triploid and this was defined basically no endosperm development. through our preliminary study using FCSS. Thus, Previous studies, such as those in Citrus, found that endosperm development is pollen dependent and all nucellar embryos originate from a single cell. Embryo endosperm formation in hickory is derived from initial cells first form thick walls, which isolate them pseudogamy. Endosperm development was essential to from the surrounding maternal tissue, and then, in later the nucellar embryo, although the endosperm did not stages, the cell walls become thinner in some of the initial supply nutrition during the early development of nucellar cells and embryogenesis becomes asynchronous embryos because the endosperm was in a rapid stage of (Koltunow et al. 1995b). In hickory, it was shown that growth. Therefore, the nucellar embryo must be the nucellus was absorbed, and only several layers of dependent upon the nucellus as a direct source of epidermal cells were left, except at the nucellar beak nutrients (Wakana and Uemoto 1987, 1988, Koltunow region. Nucellar cells in this region showed et al. 1995b). Similar to the findings in pecan (McKey morphological changes and did not have obvious 1947), the beginning of shell hardening at the micropylar boundaries between each other. Later, the whole nucellar end is an important turning point. Endosperm was in a cell mass initials formed nucellar embryos, suggesting rapid growth stage and the nucellus was absorbed that the nucellar embryos in hickory might not originate completely in early July. The developing nucellar embryo from just a single cell. The form of the nucellar embryony initial cells and proembryos used the degenerated is distinct in hickory compared to that seen in Citrus nucellus as the source of nutrition. (cluster and bud-like protuberance) (Bicknell 2004) and

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